Cole J L, Carroll S S, Blue E S, Viscount T, Kuo L C
Department of Antiviral Research, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
J Biol Chem. 1997 Aug 1;272(31):19187-92. doi: 10.1074/jbc.272.31.19187.
Ribonuclease L (RNase L) is an endoribonuclease that is activated upon binding of adenosine oligomers linked 2' to 5' to cleave viral and cellular RNAs. We recently proposed a model for activation in which activator A binds to monomer, E, to form EA, which subsequently dimerizes to the active form, E2A2 (Cole, J. L., Carroll, S. S., and Kuo, L. C. (1996) J. Biol. Chem. 271, 3978-3981). Here, we have employed this model to define the equilibrium constants for activator binding (Ka) and dimerization of EA to E2A2 (Kd) by equilibrium analytical ultracentrifugation and fluorescence measurements. Multi-wavelength sedimentation data were globally fit to the model above, yielding values of Ka = 1.69 microM and Kd = 17. 8 nM for 2',5'-linked adenosine trimer. Fluorescent conjugates of 2',5'-linked adenosine trimer with 7-hydroxycoumarin have been prepared. The coumarin emission anisotropy shows a large increases upon binding to RNase L. Analysis of anisotropy titrations yields values of Ka and Kd close to those obtained by sedimentation. The sedimentation parameters for unmodified 2',5'-linked adenosine trimer also agree with those obtained by enzyme kinetic methods (Carroll, S. S., Cole, J. L., Viscount, T., Geib, J., Gehman, J., and Kuo, L. C. (1997) J. Biol. Chem. 272, 19193-19198). Thus, the data presented here clearly define the energetics of RNase L activation and support the minimal activation model.
核糖核酸酶L（RNase L）是一种内切核糖核酸酶，在与2'-5'连接的腺苷寡聚物结合后被激活，以切割病毒和细胞RNA。我们最近提出了一种激活模型，其中激活剂A与单体E结合形成EA，随后二聚化为活性形式E2A2（Cole, J. L., Carroll, S. S., and Kuo, L. C. (1996) J. Biol. Chem. 271, 3978 - 3981）。在此，我们采用该模型通过平衡分析超速离心和荧光测量来定义激活剂结合的平衡常数（Ka）以及EA二聚化为E2A2的平衡常数（Kd）。多波长沉降数据全局拟合上述模型，对于2'-5'连接的腺苷三聚体，得到Ka = 1.69 μM和Kd = 17.8 nM的值。制备了2'-5'连接的腺苷三聚体与7-羟基香豆素的荧光共轭物。香豆素发射各向异性在与RNase L结合时大幅增加。各向异性滴定分析得到的Ka和Kd值与沉降法得到的值接近。未修饰的2'-5'连接的腺苷三聚体的沉降参数也与酶动力学方法得到的参数一致（Carroll, S. S., Cole, J. L., Viscount, T., Geib, J., Gehman, J., and Kuo, L. C. (1997) J. Biol. Chem. 272, 19193 - 19198）。因此，本文给出的数据清楚地定义了RNase L激活的能量学，并支持最小激活模型。
